Abstract
Peroxiredoxin 2 (Prdx2) is readily glutathionylated, and glutaredoxin-catalyzed deglutathionylation represents an alternative pathway for regenerating the protein's reduced form, distinct from the thioredoxin/thioredoxin reductase system (Peskin et al., JBC 216, 3053, 2016). To clarify the underlying mechanism of Prdx2 glutathionylation, we conducted kinetic analyses using stopped-flow, SDS-PAGE, and mass spectrometry-based product characterization. Kinetic modeling indicates that the reaction between Prdx2 disulfide and physiologically relevant glutathione (GSH) concentrations occurs over seconds to minutes, initially at one active site, generating glutathionylated dimers linked by a single disulfide. Further exchange with GSH leads to glutathionylation at both the peroxidatic (CP) and resolving cysteines (CR), the former predominating. We determined rate constants of 1.5 M⁻¹s⁻¹ and 0.021 s⁻¹ for thiol-disulfide exchange-mediated glutathionylation and deglutathionylation, respectively. Similar reactions subsequently occur at the second active site. The reaction rate of GSH with wild-type Prdx2 CP sulfenic acid (k = 10 M⁻¹s⁻¹) is 8–30 fold slower than when CR is mutated to Ser, Trp, or Asp, and that reaction cannot effectively compete with intramolecular condensation. Consequently, upon reaction of reduced Prdx2 with H₂O₂ in the presence of GSH, the primary product is predominantly the Prdx disulfide, and glutathionylation then follows through exchange. However, glutathionylation at CR in the presence of H₂O₂ promotes CP sulfenic acid condensation with GSH, forming diglutathionylated species and inhibiting hyperoxidation. This displaces equilibrium and accelerates Prdx2 conversion into monomeric forms. These findings help understanding the mechanism of relays between Prdx2 and other thiol proteins.
Oral presentation.